The overall goal of this project is the development of biostable organic radicals as contrast agent (CA) for magnetic resonance imaging (MRI). Such metal-free, organic-based CAs will provide a breakthrough in contrast-enhanced MRI while avoiding the risk associated with toxic metal ions. While the agents would especially benefit patients with impaired kidney function, who are at increased risk of developing nephrogenic systemic fibrosis (NSF) following administration of paramagnetic gadolinium chelates (GBCAs) for MRI procedures, the new agents may also benefit the general population. The objective of the proposed work is to synthesize organic radical contrast agents (ORCAs) that provide high quality in vivo MR images. The long-standing obstacle in the development of a practical ORCA for MRI is the design and synthesis of paramagnetic organic compounds of moderate molecular size that possess sufficiently long in vivo lifetime, high 1H water relaxivity (r1), and high water solubility. We propose a design strategy to prepare ORCAs from nitroxide radicals that are highly resistant to reduction and exploit prior knowledge about dendrimers and PEGylation, particularly their applications to drug design, to achieve agents with optimized MRI properties. The proposed project has three specific aims. Specific aim 1: optimize 1H water relaxivity r1 (at 3 Tesla) and hydrophilicity of ORCAs to achieve molecular r1 = 10 mMs, at least twice that of the clinical CAs (molecular r1 ??5 mM?s?). The ORCAs are derived from nitroxides conjuated to a generation 4 polypropylenimine (PPI-G4) dendrimer through PEGylation with long linear and/or branched PEG chains. The proposed work will provide a foundation for specific aim 2: optimize molecular size of the ORCAs with molecular r1 = 10 mM?s? to achieve agents with th radii 1 nm ? rSE ? 2 nm that would undergo efficient renal filtration/excretion. In vitro toxicty and water relaxivity of the ORCAs, as well as the properties that affect molecular r1, such as rotational correlation time and electron spin relaxation time, will be determined, to identify the four best ORCAs. Specific aim 3: evaluate effectiveness of ORCAs for in vivo MRI. Lowest effective dose, time course, and tumor vascular permeability (transfer constant Ktrans) for the four selected ORCAs will be determined at the clinical field of 3 Tesla by MRI of normal mice and by dynamic contrast enhanced (DCE) MRI of mice bearing tumor xenografts. Preliminary toxicity of the ORCA will be evaluated by histological examination. The most effective ORCA will be selected, labeled with a fluorescence probe, and then subjected to ex vivo biodistribution studies by electron paramagnetic resonance and fluorescence spectroscopy. These studies will determine suitability of ORCA for further development towards human use. Development of metal-free MRI contrast agents based on biostable organic radicals will expand the frontier of biomedical imaging. Modularity of organic synthesis enables efficient structural modifications for specific property tuning (redox, solubility, and toxicity). Such organic-based agents will improve early detection and diagnosis of diseases.